FRAP and FLIP analysis of MDMs expressing PH-GFP. MDMs were nucleofected to express PH-GFP and FRAP or FLIP analysis was performed 24 hours later using an UltraVIEW Vox spinning disc confocal system. Images were recorded at 6 frames/minute with a 100× Nikon oil immersion objective (NA 1.4) with Volocity 5.3.2 (Perkin Elmer). (A) FRAP: The PH-GFP-labeled IPMC (white selection) was bleached with a 20 millisecond laser pulse (488-nm laser at 50% intensity), and recovery of fluorescence was measured for 20 seconds by collecting frames at maximum speed. Data are shown in the graph on the right. (B) FLIP: The cell surface plasma membrane (white selection) was repeatedly bleached with 10 × 20 millisecond pulses of the 488 nm laser at 50% intensity using the UltraVIEW PK device. After each bleaching pulse, the fluorescence was monitored at various areas of interest for 10 seconds at maximum speed. The bleach and recovery cycle was repeated 10 times. The graph on the right shows the levels of fluorescence at the cell surface plasma membrane (PM, the photobleached area) or at the IPMC, compared to the plasma membrane of a different cell away from the bleached region (negative control). All experiments were repeated on cells from at least two donors. (C) Comparison of FRAP at the cell surface over IPMC membranes. Selected 2 × 2 μm2 areas at the cell surface or over IPMCs were photobleached, and FRAP was measured. The graph shows the average of 10 measurements in different cells from the same donor. Scale bars = 10 μm.